Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Summary

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Abstract

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

Introduction

One the most common forms of hereditary mental impairment in females, Rett syndrome, is caused by heterozygous mutations in MeCP2, an X-chromosome gene that encodes a protein essential for normal neuronal function¹. A potential approach to treating this disorder would be reactivation of the wild-type MeCP2 allele on the Xi, as restoration of MeCP2 expression was shown to reverse neurological deficits in a mouse model of this disease^1,2,4. However, epigenetic silencing of one of the two X chromosomes in female cells is tightly....

Protocol

All steps involving animals were carried out using protocols approved by the Fred Hutchinson Cancer Research Center Institutional Animal Care and Use Committee (IACUC). No reagents used here are known to pose significant human health risks except for 5-azacytidine (5-AZA).

1. Generating a reporter cell line with MeCP2-LUC-HR transgene on the Xi

1. Preparing mouse tendon fibroblasts from a female X^MeCP2-LUC-HR /X^MeCP2 mouse
   1. Euthanize a 10-12 day-old female X^MeCP2-LUC-HR/X^MeCP2 mouse.
   2. Using straight dissection scissors, make a circumferential....

Results

Mouse tendon fibroblasts were harvested from a previously described X^MeCP2-LUC-HR/X^MeCP2 female mouse, immortalized by retroviral transduction of E6 and E7 oncogenes from HPV-16 virus7, cloned using limiting dilution, and tested for luciferase activity and expression of wild-type MeCP2 protein (Figure 1A and 1B). Luciferase activity was robust if the reporter was on Xa, and undetectable if on Xi; the native MeCP2 expression exhibited a reciprocal patter.......

Discussion

In our recent study⁶, we generated a murine cell line with luciferase and hygromycin resistance genes fused to MeCP2 on the Xi, and transduced it with a library of >60,000 shRNAs targeting >25,000 genes. We found 30 genes whose knock-down conferred survival advantage under hygromycin B selection, suggesting their role in control of MeCP2 and X-chromosome silencing. These results were validated by transducing the reported cell line with individual hairpins and assessing re.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
293FT Cell Line	Invitrogen	R700-07
5-Azacytidine	Sigma	A2385-100MG
Agencourt AMPure XP	Beckman-Coulter	A63881
Anti-MECP2 antibody	Millipore	07-013
Collagenase	Sigma	C2674-1G
Corning 96-Well Solid White Polystyrene Microplates	Fisher Scientific	07-200-336
Dulbecco's Modified Eagle Medium	Gibco	11965-092
Expression Arrest microRNA-adapted Retroviral Vector (pMSCV)	Open Biosystems	EAV4679
Expression Arrest pSM2 Retroviral shRNAmir library	Open Biosystems	RMM3796
Fetal Bovine Serum	Fisherbrand	03-600-511
HiSeq 2500	Illumina	SY–401–2501	Or equivalent
Hygromycin B	Calbiochem	400051-1MU
Lipofectamine 2000	Invitrogen	11668-019
Bright-Glo Luciferase Assay System	Promega	E2610	Homogenous Assay for Screening Colonies
Luciferase Assay System	Promega	E4530	Non-Homogenous Assay for Testing Individual Hairpins
Luminometer TopCount NXT	Perkin Elmer	N/A	Or similar luminometer
MiSeq Reagent Kit v2	Illumina	MS-102-2001	Use kit compatible with your equipment
Opti-MEM	Gibco	31985-070
Penicillin-Streptomycin	Gibco	15140-122
pMD2.G	Addgene	#12259	VSV-G envelope vector
Polybrene	Sigma	TR-1003-G
Polyethylenimine	Polysciences	23966-1
psPAX2	Addgene	#12260	Packaging vector
Puromycin	Gibco	A11138-02
Trypsin-EDTA (0.05%), phenol red	Gibco	25300-054